Rotaviruses (RVs) are significant viral pathogens that cause life-threatening diarrheal disease in infants and young children worldwide. A fascinating aspect of RVs is that they are viruses that utilize the endoplasmic reticulum (ER) for maturation. RVs exhibit a novel morphogenetic pathway that involves nascent subviral particle exit from sites of RNA replication (viroplasms) followed by particle budding through the ER membrane. Transiently enveloped particles appear in the lumen of the ER. The transient envelope is lost in a process involving rearrangement and assembly of the outer capsid proteins. The budding process involves interaction of the nascent subviral particles with the unique RV non-structural protein 4 (NSP4) that functions as an intracellular receptor in the ER membrane. Other remarkable properties of NSP4 include its being the first described viral enterotoxin. Thus, NSP4 has several roles in RV replication and morphogenesis, processes that are novel, complex and poorly understood. These data also highlight the possibility of using NSP4 and RV maturation to discover new mechanisms of cell function and as targets for inhibiting viral replication. The studies proposed in this new, revised grant seek to understand the role(s) of NSP4 in modulating cellular calcium signaling as a mechanism to regulate virus replication and morphogenesis. Our long term goal is to elucidate the mechanisms by which this novel protein controls virus replication and to target these mechanisms to attenuate disease. The central hypothesis of our proposed research is that NSP4 is a key regulator of rotavirus replication and morphogenesis. We predict that the abilities of NSP4 to alter intracellular calcium homeostasis, to modulate ER membrane and viroplasm localization, and to interact with the stalk domain of the capsid spike and VP6 on double-layered particles result in molecular switches that regulate viral morphogenesis. The specific aims of our proposed work are (1) To determine how NSP4 affects calcium homeostasis, and (2) To dissect NSP4 regulation of viral morphogenesis. Because RVs mature in the ER and exit cells using a nonclassical vesicular pathway that is Golgi-independent, we expect to elucidate new mechanisms of complex virus assembly, including new insights into the autophagy pathway and RV subversion of calcium-regulated processes in polarized intestinal cells. These studies are significant and of fundamental interest because intracellular membrane perturbation is not well understood for any non-enveloped virus and several such viruses enter or alter the ER as part of their replication cycles. In addition, modulation of levels of intracellular calcium and intersection with cellular autophagy are important pathways exploited by many viruses. PUBLIC HEALTH RELEVANCE: The studies proposed in this revised grant seek to understand the role(s) of NSP4 in modulating cellular calcium signaling as a mechanism to regulate virus replication and morphogenesis. Our long term goal is to elucidate the mechanisms by which this novel protein controls virus replication and to target these mechanisms to attenuate disease. The specific aims of our proposed work are (1) To determine how rotavirus NSP4 affects calcium homeostasis, and (2) To dissect NSP4 regulation of viral morphogenesis.